An efficiently operating haemostatic system is of vital necessity for the mammalian organism. In healthy organisms defects of the blood vascular system, e.g. vascular lesions, are repaired in a two-step process: the aggregation of thrombocytes is followed by the formation of a fibrin clot in an enzyme cascade under participation of several blood clotting factors. Most of these factors are serin proteases, for example thrombin which catalyzes the reaction of fibrinogen to fibrin. The coagulation system is counteracted by the fibrinolytic system involving, among others, the protease plasmin which cleaves fibrin. The coagulation and fibrinolytic systems are usually in a dynamic equilibrium. In cases, however, in which the fibrinolytic potential of the organism is disturbed or insufficient, for example in patients suffering from thromboembolisms or post-operative complications, it is indispensable to support the organism by the administration of anticoagulants to prevent further formation of fibrin and of thrombolytic agents to dissolve the formed thrombi.
Hirudin, an anticoagulant that occurs naturally in leeches (Hirudo medicinalis), is a potent and specific inhibitor of thrombin, preventing the cleavage of fibrinogen and subsequent fibrin clot formation. Hirudin reacts very rapidly with .alpha.-thrombin to form a very tight noncovalent complex (K.sub.I .apprxeq.1-0.01 pM) which is extremely stable and enzymatically totally inactive. Several closely related hirudin variants have been described, each containing 65 or 66 amino acids, for example the variants designated hirudin variant 1 (HV1), hirudin variant 2 (HV2), hirudin variant PA (HV3), and "des-(Val).sub.2 -hirudin". The variants differ from each other by a number of amino acids, but all have an accumulation of hydrophobic amino acids at the N-terminus, an accumulation of polar amino acids at the C-terminus, a tyrosine residue (Tyr 63) present as sulphate monoester, three disulphide bridges and the anticoagulant activity in common. Recently, cDNAs and synthetic genes coding for hirudin variants have been cloned and expressed in microbial hosts. The recombinant hirudin variants lack the sulphate monoester group at Tyr 63 and are therefore also referred to as desulphatohirudins. However, they exhibit biological properties at least equivalent to those of natural sulphated hirudins.
Hirudin has a great potential for future therapeutic use due to its selective inhibition of thrombin in conjunction with its low toxicity and the absence of immunological side effects. However, the successful therapeutic application of hirudin also requires a system for monitoring its activity as well as the course of the therapy. In addition, it is desirable to be able to determine the actual requirement for hirudin treatment. A solution to these problems would be the development of antibodies against the complex formed by thrombin and hirudin which could be employed to detect the formation of thrombin even in small amounts.